The precursor of the F-1 beta subunit of the ATP synthase is covalently modified upon binding to plant mitochondria

We present evidence for a unique covalent modification of a nuclear-encoded precursor protein targeted to plant mitochondria. We investigated the earl y events of in vitro import for the mitochondrial precursor of the ATP synt hase F(1)beta subunit from Nicotiana plumbaginifolia (pF(1)beta) into plant mitochondria. When pF(1)beta of 59 kDa was incubated with mitochondria iso lated from different higher-plant species, a band of 61 kDa was generated. The 61 kDa protein was a covalently modified form of the 59 kDa pF(1)beta. The modification was dependent on the 25 amino acid long N-terminal region of the presequence of pF(1)beta. The modification was catalysed by an enzym e located in the outer mitochondrial membrane which was specific for higher plants and could not be washed off from the membrane by urea, KCl or EDTA. The modification was ATP- and Ca2+-dependent, but it was not affected by i nhibitors of protein kinases. No inhibition of the modification was observe d with phosphatase, methylation or acylation inhibitors. The modification o ccurs prior to translocation through the mitochondrial outer membrane. Inhi bition of the modification process does not affect the import of the precur sor protein, hence precursor modification was not a prerequisite for import . Both the modified and the unmodified pF(1)beta proteins were strongly ass ociated with the mitochondrial outer membrane.